Green Chemistry This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Metal-free cysteamine-functionalized graphene alleviates mutual interferences in heavy metal Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. Cite this: Green Chem., 2023, 25, 1647 electrochemical detection† Qiuyue Yang, a,b Emily P. Nguyen,a David Panáček,a,c Veronika Šedajová, c Vítězslav Hrubý,c,d Giulio Rosati,a Cecilia de Carvalho Castro Silva, a,e Aristides Bakandritsos, c,f Michal Otyepka c,g and Arben Merkoçi *a,h Heavy metal pollutants are of great concern to environmental monitoring due to their potent toxicity. Electrochemical detection, one of the main techniques, is hindered by the mutual interferences of various heavy metal ions in practical use. In particular, the sensitivity of carbon electrodes to Cd2+ ions (one of the most toxic heavy metals) is often overshadowed by some heavy metals (e.g. Pb2+ and Cu2+). To mitigate interference, metallic particles/films (e.g. Hg, Au, Bi, and Sn) typically need to be embedded in the carbon electrodes. However, these additional metallic materials may face issues of secondary pol- lution and unsustainability. In this study, a metal-free and sustainable nanomaterial, namely cysteamine covalently functionalized graphene (GSH), was found to lead to a 6-fold boost in the Cd2+ sensitivity of the screen-printed carbon electrode (SPCE), while the sensitivities to Pb2+ and Cu2+ were not influenced in simultaneous detection. The selective enhancement could be attributed to the grafted thiols on GSH Received 10th August 2022, sheets with good affinity to Cd2+ ions based on Pearson’s hard and soft acid and base principle. More Accepted 13th January 2023 intriguingly, the GSH-modified SPCE (GSH-SPCE) featured high reusability with extended cycling times DOI: 10.1039/d2gc02978b (23 times), surpassing the state-of-art SPCEs modified by non-covalently functionalized graphene deriva- rsc.li/greenchem tives. Last, the GSH-SPCE was validated in tap water. 1. Introduction Heavy metal (HM) pollutants influence important access to a clean water due to the risks of toxicity, bioaccumulation, bio- Nanobioelectronics and Biosensors Group, Catalan Institute of Nanoscience and magnification, and environmental persistence.1,2 On the Nanotechnology (ICN2), CSIC, Campus UAB, Bellaterra, Barcelona 08193, Spain. E-mail: arben.merkoci@icn2.cat Environmental Quality Standards Directive List, As, Cd, Cr, Cu, b Department of Materials Science, Universitat Autònoma de Barcelona, Campus de la Fe, Ni, Pb, Hg, and Zn are underlined as key substances for UAB, Plaça Cívica, 08193 Bellaterra, Barcelona, Spain evaluating water quality.3 Although some HM elements (e.g. c Regional Centre of Advanced Technologies and Materials, Czech Advanced Cu) are necessary for human health, ingestion of these HMs is Technology and Research Institute (CATRIN), Palacký University Olomouc, Šlechtitelů harmful at high concentrations4,5 while other HMs, such as 27, 783 71 Olomouc, Czech Republic d Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc, Cd and Pb are harmful even at the ppb range to the ecosystem 17. listopadu 12, 771 46 Olomouc, Czech Republic and bioaccumulate in the human body via the foods we eat.5 e MackGraphe-Mackenzie Institute for Research in Graphene and Nanotechnologies, Therefore, HM analysis within primary food sources (e.g. Mackenzie Presbyterian University, Consolação Street 930, 01302-907 São Paulo, drinking/tap water) is important. Brazil f Given their simplicity and low cost, electrochemical tech- Nanotechnology Centre, Centre of Energy and Environmental Technologies, VŠB– Technical University of Ostrava, 17. listopadu 2172/15, 708 00 Ostrava-Poruba, niques are ideal for HM ion analysis. Amongst these, square- Czech Republic wave anodic stripping voltammetry (SWASV) is favourable due g IT4Innovations, VSB–Technical University of Ostrava, 17. listopadu 2172/15, 708 00 to its resilience to dissolved oxygen in real samples.6,7 When Ostrava-Poruba, Czech Republic determined by SWASV, HM cations are first reduced on the h Institució Catalana de Recerca i Estudis Avançats, Pg. Lluís Companys, 23, working electrode surface at a negative potential over a certain Barcelona 08010, Spain † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ period of time (in minutes); then the potential is increased to 10.1039/d2gc02978b a positive value with a ramp upon which a square waveform is This journal is © The Royal Society of Chemistry 2023 Green Chem., 2023, 25, 1647–1657 | 1647 View Article Online Paper Green Chemistry superimposed; moreover, reduced HMs are oxidized to the modify the SPCE and reported an enhanced sensitivity of the corresponding cations, generating discrete anodic current SPCE to Cd2+ with the presence of Hg2+, Cu2+, and Pb2+.33 peaks. Consequently, the corresponding peak potential allows However, the disposal device could not operate under continu- the identification of HM species, and the peak intensity or ous measurement, which is the final hurdle to overcome. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. area is proportional to the HM concentration. Hence, reusable biofunctionalized nanomaterials are highly In the field of HM detection by SWASV, carbon electrodes sought. have gradually replaced the hanging mercury drop electrode Graphene as a 2D nanomaterial is a good host for biofunc- (HMDE) due to the toxicity of Hg.8 However, bare carbon elec- tionalization due to the large basal area.34 The functionali- trodes suffer from mutual interference due to the lack of an zation of graphene is primarily divided into non-covalent and effective working surface in comparison to HMDEs. This could covalent functionalization.35 Non-covalent functionalization Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. induce ion competition and intermetallic compounds in the takes advantage of the π-interactions, hydrogen bonding, van deposition step, which are generally regarded as two major der Waals force, etc. between graphene and functionalization reasons for mutual interference.8–10 Mutual interference often agents, while covalent functionalization creates a more robust results in unexpected outcomes, including a drop in sensi- chemical bond between the two. Thus, covalent moieties are tivity, shifting of the peak potential, peak splitting, and peak extremely stable on the graphene surface.36 Accordingly, co- overlapping.11 In particular, the sensitivity to Cd2+ is dimin- valently biofunctionalized graphene derivatives seem promis- ished by the presence of Pb2+ or Pb2+ and Cu2+ when deter- ing to address mutual interferences while maintaining high mined using screen-printed carbon electrodes (SPCEs),12,13 stability and reusability. Nevertheless, the most commonly glassy carbon electrodes (GCE),14 or boron-doped diamond used precursors, GO and reduced graphene oxide (rGO), pose electrodes (BDDs).15 Mutual interference makes the detection challenges to homogeneous and manageable functionalization of Cd2+ incredibly challenging with the existence of Pb2+ and due to their non-uniform distribution of oxidized groups.37,38 Cu2+ ions, which commonly appear in many waters. Solving As such, a new methodology for functionalizing graphene this issue is more daunting for SPCEs as their rough surface covalently has been investigated,39 in which fluorographene tends to induce mutual interference compared to other carbon (FG) is used as a precursor with uniformly distributed fluorine. electrodes. Controllable graphene chemistry can be accessed through the The most typical alleviating strategy is to deposit metallic nucleophilic substitution reaction on the FG and the simul- substances (e.g. Bi,16 Hg,17 Sn,18 and Au19) on carbon electro- taneous defluorination.40 For example, it was reported that car- des to mimic the HMDE.20 Despite the effectiveness of this boxylic groups could be uniformly and covalently functiona- strategy, most of these metals are scarce in the earth’s crust, lized on graphene sheets (and to form graphene acid, GA) via which renders them unsustainable, together with recycling the mild hydrolysis of cyanographene, which is a practically issues, while the mining and smelting may cause secondary fluorine-free graphene derivative with covalently bonded pollution to the environment.21 Amongst them, on account of nitrile groups synthesized from FG.41 The carboxyl groups on its moderate cost and low toxicity, Bi, generally regarded as a the GA then behave as versatile anchoring sites to conjugate “green metal”, is one of the most favoured ones,22 although with many biomolecules. These formed derivatives have been recently the greenness of some Bi complexes has been utilized in versatile applications, like energy storage,42 doubted.23 Besides, owing to the working principle of metallic sensing,43 and catalysis.44 More excitingly, since organic electrodes forming alloys with HM ions in the deposition step, amines commonly appear in biomaterials and are known to be the electrodes tend to lose their component substances in the good nucleophiles,45 they can react with FG, affording formed alloys during stripping, thus resulting in only being materials with molecules grafted on the graphene backbone able to be reused a relatively few times (∼10 times),24–26 which directly via the covalent bonds of amino groups with the gra- could limit their application with the developments of in situ phene surface. This covalent functionalization strategy can and automatic sensing tools used for water quality control in open the way to facile and flexible functionalization with large lakes and rivers recently.27 Furthermore, emerging 2D various biomolecules. materials (e.g. Ti3C2) and porous materials (e.g. ZIF-8) are This study is, to the best of our knowledge, the first to employed to enhance Cd2+ sensitivity under mutual report the synthesis of a cysteamine-functionalized graphene interference.28,29 In a bigger scope, general mutual inter- (GSH) by this covalent functionalization strategy and then its ference can be reduced by the use of other nano-structured modification on the screen-printed carbon electrode (SPCE), metal compounds.30–32 However, the synthesis of these with an aim to solve mutual interferences without requiring materials relies on reactions involving metal salts as precur- any metallic additives. By leveraging the binding proclivity of sors, which induces similar problems. Therefore, green and thiol residues on the GSH towards the softer Cd2+ ions based stable nanomaterials are highly demanded in order to alleviate on Pearson’s hard and soft acid and base principle (HSAB), the mutual interference issues. GSH was expected to enhance the sensitivity of the SPCE to Fortunately, bio- and carbon-based nanomaterials rich in Cd2+. Hence, the role of the GSH was examined for simul- N, S, and O also appear to be able to address the issue due to taneous HM ions (i.e. Cd2+, Pb2+, and Cu2+) detection in a flow their good affinity towards specific HM ions. Choi et al. uti- injection system designed for in situ and automatic measure- lized graphene oxide (GO)-doped diaminoterthiophene to ments (Fig. 1a and Fig. S1†) based on SWASV (Fig. 1b). 1648 | Green Chem., 2023, 25, 1647–1657 This journal is © The Royal Society of Chemistry 2023 View Article Online Green Chemistry Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. Fig. 1 Schematic illustration of the working principle in our study. Schematic illustration of the (a) flow injection system used for in situ HM detec- tion, allowing the sample and supporting electrolyte to mix automatically, and to flow through the surface of the SPCE in the deposition step and to stop in the stripping step during SWAVS; (b) two main two of SWASV: the deposition and stripping steps; and (c) use of GSH for Cd2+-sensing enhancement compared with a bare SPCE by the functionalized cysteamine moieties and graphene-like 2D structure. Besides, the GSH was benchmarked against other graphene smoothed and baseline corrected for better clarity. GSH was derivatives functionalized with other groups, verifying its characterized by the XPS technique (PHI VersaProbe II physical superior sensitivity to Cd2+ (Fig. 1c). Furthermore, continuous electronics spectrometer) using an Al Kα source (15 kV, 50 W), cycling measurements were tested by using the GSH-SPCE on a MultiPak system (Ulvac-PHI, Inc.). A software package was with the expectation of a greater sensing reusability due to the used to evaluate the obtained data. Raman analysis was oper- covalent functionalization of the thiol groups on the graphene ated on a DXR Raman microscope using a diode laser’s surface. Last, the GSH-SPCE was used to for detection in real 633 nm excitation line. The spiked tap water was analysed in a samples. 7500ce inductively coupled plasma mass spectrometry (ICP-MS) instrument (Agilent). 2.2 Synthesis of the GSH 2. Experimental section First, 2 g of graphite fluoride (61 < at% F, polymer, Sigma- 2.1 Reagents and equipment Aldrich) was dispersed in 120 ml of DMF (for peptide syn- The SPCEs were fabricated with a DEK248 printer machine thesis, Merck), followed by continuous stirring for 72 h at (DEK, Weymouth, UK). The Ag/AgCl ink was Loctite EDAG room temperature. Then, the dispersion was sonicated in an AV458 (Henkel). The carbon paste was C2030519P4 CARBON ultrasonication bath for 4 h (<60 °C) and was kept stirring for SENSOR PASTE (267508), the Ag ink was C2180423D2 SILVER another 24 h at room temperature. Last, 10.2 g K2CO3 (Penta) PASTE 349288, and the insulating ink was D2070423P5 and 5 g cysteamine (Sigma-Aldrich) were added to the dis- DIELECT PASTE GREY (all from Sun Chemical). Deionized persion and left stirring under heating at 130 °C for 24 h in an water (18.2 MΩ·cm at 25 °C, Milli-Q) was used throughout for oil bath in the hood with a condenser for reflux. all the experiments. The 37% hydrochloric acid was 320331- When cooling down, the formed product was centrifuged 2.5L from Sigma. Standard HM solutions (Cd2+, Cu2+, and Pb2+ (20 000 rcf per 10 min) and washed with DMF (2×), hot DMF 1000 ppm, Sigma) were all AAS grade. GO water solution (1×), acetone (2×), hot acetone (1×), ethanol (3×), distilled water (N002-PS-1.0) was acquired from Angstron Materials (Dayton, (2×), and hot distilled water (1×). OH, USA). To decompose the possible unwanted disulfide bonds in The concentration of graphene supernatants was estimated the GSH, the whole amount of the synthesized material was by UV–VIS spectrophotometry (UV-1900, Shimadzu). The mor- mixed with 420 µL of 2-mercaptoethanol (Carl Roth; ∼2× phology of the material was characterized by SEM analysis molar ratio of –SH groups considered as 20% F.D.), and left with a Hitachi SU6600 instrument with an accelerating voltage shaking for 30 min. Then, the material was additionally of 5 kV, and by TEM analysis with a JEM 2010 TEM instrument washed with ethanol (1×), acidified distilled water (2×), and (Jeol, Japan). The FTIR characterization was operated on an iS5 distilled water (1×) using centrifugation (20 000 rcf per 10 min) FTIR spectrometer (Thermo Nicolet), equipped with a Smart and purified by being placed into the dialysis membrane (cut- Orbit ATR accessory with ZnSe crystal. FTIR spectra were off 14 kDa) in a container of distilled water (5L) that was This journal is © The Royal Society of Chemistry 2023 Green Chem., 2023, 25, 1647–1657 | 1649 View Article Online Paper Green Chemistry changed every day until the dispersion containing the material Microsoft Excel. Recovery was determined as the tested con- reached a conductivity <100 µS cm−1. centration divided by the spiked concentration.48 2.3 Preparation of GSH-SPCEs This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. SPCEs were fabricated the same as reported in our previous 3. Results and discussion study.27 For better quality control, all the SPCEs were cleaned 3.1. Synthesis and characterizations of the GSH in 0.05 M HCl and deionized water, and then pre-tested by SWASV. According to the obtained stripping curves (charge vs. The GSH was synthesized through the substitution of F on potential), the SPCEs were selected only within the charge fluorographene (FG) by the amino groups (–NH2) on cystea- range of 2–4 mC. Additional cyclic voltammetry (10 scans) was mine (Fig. 2a). The defluorination and functionalization Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. performed with the selected SPCEs in 0.05 M HCl for further occurred simultaneously owing to the nucleophilic substi- cleaning. tution.49 To secure the deprotonation of the amino groups A suspension of the graphene derivatives (GSH, GA, and (and thus their maximum nucleophilicity), potassium carbon- GO, 1 mg ml−1) was sonicated in a bath for 30 min at room ate (K2CO3) was used as a base to scavenge protons from the temperature and then centrifuged at 9000 rpm for 1 min. Next, system, preventing the formation of the by-product, hydrogen 5 μL of the GSH supernatant (∼0.016 mg ml−1) was pipetted fluoride (HF), during the synthesis. Additionally, K2CO3 onto the working electrode of a cleaned SPCE and dried at boosted the surface area of the resulting material thanks to 37 °C in an oven. Depending on the total modified volume (10, the release and expansion of carbon dioxide.50 15, and 20 μL), the drop-casting procedure was repeated The SEM and TEM images in Fig. 2(b–d) demonstrate several times in 5 µL increments. Other graphene derivatives typical 2D sheets of functionalized graphene with a wrinkled (GA and GO) supernatants followed the same approach, except morphology. their concentrations were adjusted equal to that of the GSH The FTIR results showed that the GSH was a fluorine-free supernatant. All the supernatant concentrations were esti- graphene-based material, due to the absence of a sharp feature mated by UV–Vis using the Beer–Lambert equation, whereby at 1200 cm−1 originating from C–F bond vibration, while this the molar absorptivity was 3463 ml mg−1 m−1.46 The GO-SPCE feature was apparent in the pristine graphite fluoride (GF) was reduced (to rGO-SPCE) in the cyclic voltammetry tests (CV, material (Fig. 2e). The development of sp2 regions was evident 10 scans) between −1.1 and 0 V, then by chronopotentiometry from the strong CvC band at 1550 cm−1. The broad feature in at −1.1 V for 200 s. the region between 1300 and 1050 cm−1 is common for many graphene-based materials, representing a large group of 2.4 HM detection by SPCEs vibration modes whose bands are mutually indistinguishable. The largest part of the features represented graphene’s in- HM analysis was conducted in the fluidic injection system plane C–C bond stretching,41 while C–S,51,52 C–N,53 and C–O54 shown in Fig. 1 and as per our previous study.27 Fig. S1† species of the immobilized cysteamine molecules and oxygen describes the working principle of this system in detail. The contamination may also have contributed to its intensity and frequency of the square waves was 25 Hz with an amplitude of broadness. Last, among this broadband, two sharp maxima at 30 mV and a potential step of 6 mV. The equilibrium time was 1123 and 1023 cm−1 were distinguishable. The shoulder at 20 s. 1450 cm−1 was assigned to C–H bending, which was the only proof of the presence of C–H bonds, since the C–H stretching 2.5 Data analysis band at 2900 cm−1 was barely observable.50 Last, a broad The integral area under each peak (A) in voltammograms was feature manifested between 3050 and 3500 cm−1 was assigned used as the sensing signal instead of the peak current due to to O–H and N–H stretching vibrations.50 peak splitting, where A is the integral of the splitting peak area The elemental composition from the XPS analysis (Fig. 2f ) (two subpeaks in total), which was calculated using the soft- showed that the GSH was nearly fluorine-free (1.2 at%) con- ware PStrace 5.8 (PalmSens). The LOD was estimated based on taining C, N, and S atoms, complying with the results from the International Conference on Harmonization’s Q2 FTIR. The minimal oxygen content could either originate from Validation of Analytical Procedures by:47 the DMF molecules reacting with the GF during functionali- zation,41 or from adventitious contamination from the LOD ¼ 3σ=S environment. The functionalization degree (F.D.) was esti- where σ and S are the intercept standard error and the slope of mated to be 3.5% based on the S/C ratio.41 In the C 1s region the fitted calibration line, respectively, obtained using Origin of the XPS spectrum (Fig. 2g), carbon atoms were found 2018 software. The sensitivity (S) is defined as the calibration mainly in the sp2 hybridization state, further proving the gra- slope. Repeatability is defined as the relative standard devi- phene-like nature of the material observed from the FTIR ation (RSD) of the sensing signals from continuous measure- results, while the other components found at higher binding ments. Reproducibility is the RSD of the sensitivities collected energies corresponded to sp3-hybridized carbon atoms, mostly from different SPCEs in one batch. The t-test used was the from the alkyl chains of cysteamine functionalities and the 2-tailed Student’s t-test with heteroscedasticity conducted in carbon bonded to sulfur, nitrogen, and oxygen atoms, and to 1650 | Green Chem., 2023, 25, 1647–1657 This journal is © The Royal Society of Chemistry 2023 View Article Online Green Chemistry Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. Fig. 2 Characterizations of the GSH. (a) Schematic illustration of the synthesis. (b) Scanning electron microscopy (SEM), (c) transmission electron microscopy (TEM), and (d) magnified TEM images of the GSH sheet. (e) Fourier-transform infrared spectroscopy (FTIR) spectra of the GSH and graph- ite fluoride (GF). (f ) X-Ray photoelectron spectroscopy (XPS) survey spectrum and element composition analysis. High-resolution XPS spectra for (g) C 1s and (h) S 2p. residual fluorine with primarily overlapping binding energy tionalities and the graphene sheet, and thus, reflecting the values. high F.D. of the material. Moreover, the sharp G-band showed In the deconvolution of the S 2p envelope, the sulfur atoms that the GSH had developed a network of sp2 carbon, denoting of GSH were mostly bonded to carbon atoms, as indicated by a high degree of reductive defluorination, which also accorded the component at 163.9 eV for S 2p3/2 which further proved the with the C 1s spectrum in the XPS results, in which the carbon successful functionalization, although some sulfur atoms were sp2 hybridized atoms constituted a dominant fraction of the also bonded to oxygen atoms, as evident from the presence of GSH (Fig. 2g). additional components at higher binding energy values Overall, the characterization results allowed concluding (Fig. 2h).55 that the reaction of FG with cysteamine afforded a graphene- A broad D band and rather high ID/IG ratio (1.26) in the like material functionalized with thiol groups. This was thanks Raman spectrum (Fig. S2†) indicated that the GSH contained a to the covalent bond between the cysteamine molecules via high number of defects within the graphene backbone. This their amino groups and the graphene sheet. However, it is phenomenon should stem mainly from the sp3 carbon atoms, important to note that to further comply with green chemistry signifying the successful bonding between cysteamine func- principles, the replacement of DMF in syntheses with more This journal is © The Royal Society of Chemistry 2023 Green Chem., 2023, 25, 1647–1657 | 1651 View Article Online Paper Green Chemistry benign solvents will be required due to health-related con- could be influenced by the highly concentrated Pb2+ ions cerns. Our recent work demonstrated the possibility to achieve (Fig. 3c). this goal, showing the functionalization of fluorographene (in In the corresponding voltammograms, intriguingly, the a different reaction), which took place similarly effectively both expected Pb peak was divided into two peaks: peak A, centred This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. in DMF and in acetonitrile.56 Thus, it is anticipated that the at −0.56 V and commonly identified as the main Pb peak,57 reaction of fluorographene with cysteamine could be also furn- and peak B at −0.49 V (Fig. 3(d and e)). However, the splitting ished in acetonitrile, which will be the subject of a follow-up of peak B was unobservable when detecting only Pb2+ at the work related to a different application. same concentration using both electrodes of GSH-SPCE and bare SPCE (Fig. S9†). This suggests that it was impossible for 3.2 Sensing performance of the GSH-SPCE compared to the peak B to stem from the interaction of Pb–carbon, Pb–GSH, or Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. bare SPCE Pb–Pb, and therefore, the most plausible rationale is that it 3.2.1 Fabrication, optimization, and sensing performance was caused by Pb–Cd interactions. As reported in the previous of the GSH-SPCE in individual HM solutions. The GSH-SPCE study, the appearance of peak B could be attributed to the het- was prepared by drop casting, in which the supernatant of erogeneity of the electrode surface where multiple cations are GSH (∼0.016 mg ml−1) was pipetted on the working electrode deposited.57 A similar phenomenon was found in other of a cleaned bare SPCE. After the supernatant had completely studies too.58,59 evaporated, the procedure was repeated once more to achieve Based on the idea that peak B can act as an indicator to the best modification. evaluate the interaction of Pb and Cd, the maximum current All the following experiments were operated under the opti- of peak B was plotted with the corresponding Cd2+ concen- mized situation, i.e. HCl as a supporting electrolyte at 0.05 M, tration in Fig. 3f (red rectangle for GSH-SPCE, black rectangle pipetted GSH supernatant volume of 10 μL, and deposition for bare SPCE). The much lower value of the GSH-SPCE with potential of −1.0 V with a flowrate of 3 ml min−1 and flowtime the independence of the Cd2+ concentration proves that GSH of 200 s, as shown in Fig. S3–S5 and Table S1.† could help the SPCE to reduce the mutual interference of Cd– Bare SPCE and GSH-SPCE were first employed to detect Pb. Cd2+, Pb2+, and Cu2+ individually with different concentrations After investigating the enhancement of Cd2+ sensitivity by to determine their sensitivities (S, denoted as the slope of the GSH with the interference of Pb2+ or Cu2+ separately, the ques- calibration line). Compared to the bare SPCE, GSH-SPCE tion was aroused whether GSH could even alleviate the mutual clearly showed higher sensing signals ( peak areas) for each interference amongst the 3 HMs ions (i.e. Cd2+, Pb2+, and concentration of all the HM ions (Fig. S6(a–c)†) as expected, Cu2+). Hence, a comparative experiment between the which was compatible with the fact that GSH offered faster GSH-SPCE and bare SPCE was conducted in mixed solutions charge-transfer rates with lower charge-transfer resistance, as containing these 3 HMs ions with only one HM varying its con- shown in the cyclic voltammetry (CV) and electrochemical centration but the other two remaining at 80 ppb. In Fig. 3(g– impedance spectroscopy analysis (EIS, Fig. S7 and Table S2†). i), it can be seen that the GSH-SPCE significantly increased the 3.2.2 Detection of Cd2+ under the interference of Pb2+ and Cd2+ sensitivity and linearity with the interference of Pb2+ and Cu2+. Cd2+ is the most difficult HM to detect compared to Pb2+ Cu2+, compared to the bare SPCE, which on the other hand and Cu2+ in their simultaneous presence due to the dimin- showed a negligible sensitivity and linearity (Table S3†). ished sensitivity by the presence of the other two, as found in However, regarding the sensitivities of Pb2+ and Cu2+, both our previous study (Fig. S8†).27 This could be attributed to were minimally influenced by the GSH (Fig. 3(h and i)). It was three factors in the deposition step: The GSH-SPCE and bare noticeable that the linearity of the GSH-SPCE to detect Cd2+ SPCE were tested in 80 ppb Cd2+ with different concentrations with the existence of Cu2+ and Pb2+ was relatively low, which of Cu2+ or Pb2+ ranging from 0 to 160 ppb. The Cd signals of could limit its practical use. To solve this issue, machine learn- the GSH-SPCE only fluctuated slightly with different concen- ing using other fitting algorithms would be considered in prac- trations of Cu2+, which indicated the minimal influence from tical use.60 Cu2+ on the Cd2+-sensing signals (Fig. 3a). Regarding the 3.2.3 Simultaneous detection towards Cd2+, Pb2+, and evaluation of Pb2+ interference, the GSH-SPCE enabled obser- Cu2+. To further understand the simultaneous sensing per- vable Cd2+ signals, while the counterpart only showed unmea- formance, the GSH-SPCE was examined in a mixed solution surable signals (Fig. 3b). This proved that the GSH significantly containing multi-HMs of Cd2+, Pb2+, and Cu2+ with a fixed alleviated the Pb2+ interference on Cd2+ and allowed the simul- ratio of 1 : 1 : 1 (Fig. 4). As a comparison, the bare SPCE was taneous detection of both, which is incredibly difficult for bare tested under the same conditions. The sensitivity of the SPCEs. GSH-SPCE to Cd2+ showed a 6-fold enhancement compared to To further evaluate the influence of the Pb2+ concentration its counterpart; however, the sensitivities of Pb2+ and Cu2+ on Cd2+ sensitivity, the GSH-SPCE and bare SPCE were used to were only slightly increased by 1.5 times (Table S3†). Besides, detect various concentrations of Cd2+ in two scenarios: with the bare SPCE had poor linearity to Cd2+ (R2 = 0.84) due to the the presence of 10 ppb and 80 ppb Pb2+. In both scenarios, the influence from Pb2+ and Cu2+, while the GSH-SPCE’s linearity GSH was proved to substantially increase the sensitivity of the was comparatively much better (R2 = 0.99). Moreover, the SPCEs to Cd2+, even though the sensitivity of the GSH-SPCE GSH-SPCE had a lower LOD to Cd2+ (15 ppb) than the bare 1652 | Green Chem., 2023, 25, 1647–1657 This journal is © The Royal Society of Chemistry 2023 View Article Online Green Chemistry Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. Fig. 3 Sensing performance of the GSH-SPCE and bare SPCE to Cd2+ under the mutual interference of Pb2+ and Cu2+. Peak areas of 80 ppb Cd2+ by the GSH-SPCE and bare SPCE with various concentrations of (a) Cu2+ and (b) Pb2+ respectively. (c) Calibration curves of Cd2+ in the GSH-SPCEs and bare SPCEs with Pb2+ as an interfering ion at 10 ppb and 80 ppb. Voltammograms of the (d) bare SPCE and (e) GSH-SPCE to detect various con- centrations of Cd2+ (from 0 (black) to 160 ppb (yellow)) simultaneously with the presence of 80 ppb Pb2+. (f ) Peak B maxima of the GSH-SPCE (red rectangle) and bare SPCE (black rectangle) with varying the concentration of Cd2+. Calibrations of the bare and GSH-SPCEs towards (g) Cd2+ under the interference of 80 ppb Pb2+ and Cu2+, (h) Pb2+ under the interference of 80 ppb Cd2+ and Cu2+, and (i) Cu2+ under the interference of 80 ppb Cd2+ and Pb2+. SPCE (84 ppb), with a linear range up to 200 ppb. Regarding be more reusable (23 times) than both the non-covalently func- Cu2+ and Pb2+, the LODs showed slight improvement by GSH tionalized graphene derivatives-modified electrodes (1–10 (Table S4†). All the stripping peaks (Fig. 4d) were well separ- times) and most bismuth-based electrodes (5–16 times), ated, where the peaks at −0.86 and −0.2 V referred to Cd and showing comparable repeatability to Pb2+ (3.68%) and Cu2+ Cu, respectively, and the splitting peaks around −0.5 V could (6.69%). However, the repeatability of Cd2+ was relatively low, be identified as Pb. The splitting peak could also be attributed which would limit the GSH-SPCE’s application in the highly to the existence of Cu2+ leading to the formation of Cu–Pb precise detection of Cd2+ ions. Instead, low-cost, rapid, and intermetallic compounds.61 automatic sensing tools (e.g. an automatic in situ sensing boat 3.2.4 Reusability and reproducibility. The GSH incorporat- navigating in large natural waters) that rely on their reusability ing thiol moieties covalently bound on the graphene sheet was more than precision could be compatible with the sensing expected to be more robust and reusable than the non-co- system in this study.27 valently functionalized one. Hence, GSH-SPCE was subjected Reproducibility is pivotal for mass production and thus, to 25 continuous measurements in a mixed solution of Cd2+, five GSH-SPCEs and five bare SPCEs were tested in the same Pb2+, and Cu2+ ions. The repeatability was recorded as 28.3%, mixed solutions of Cd2+, Pb2+, and Cu2+ with the ratio of 3.86%, and 6.69% for Cd2+, Pb2+, and Cu2+, respectively. It 1 : 1 : 1. All the obtained sensitivities are shown in Table S6.† should be noted that the relatively high repeatability to Cd2+ The reproducibility of GSH-SPCE was 17.5%, 5.1%, and 10.9% resulted from the highest signals from the first two measure- to Cd2+, Pb2+, and Cu2+, respectively. ments (Fig. S10a†), which was typical for the bare SPCE, too 3.2.5 Statical analysis of GSH for enhancing Cd2+ sensi- (Fig. S10b†). Digging out the first two abnormal results, the tivity in simultaneous detection and comparison with other GSH-SPCE was reused 23 cycling times (RSD ≤ 20%). graphene derivatives. To further prove the enhancement by the Compared with other studies in Table S5† demonstrating the GSH statistically, Student’s t-tests were performed to analyse reusable times and repeatability, the GSH-SPCEs were found to all the sensitivities data obtained in the reproducibility study This journal is © The Royal Society of Chemistry 2023 Green Chem., 2023, 25, 1647–1657 | 1653 View Article Online Paper Green Chemistry phene sheet in the GSH, we modified SPCEs with other gra- phene derivatives as references, i.e. GA and rGO, with func- tional groups of –COOH and other oxygen groups, respectively. GA was characterized in our previous study,41,42 while GO This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. (Angstron Materials, USA) was electrochemically reduced on the SPCE in situ after drop casting.62 However, owing to their different hydrophilicity, the supernatants of GA, GO, and GSH were first adjusted by diluting to secure similar concentrations via the Beer–Lambert estimation by UV–Vis (∼0.01 mg ml−1, Fig. S11†), and were then pipetted on to cleaned bare SPCEs Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. with the same optimized procedure. The GSH showed the best sensitivity to Cd2+ compared to GA and rGO (after in situ reduction) in the simultaneous detec- tion towards Cd2+, Pb2+, and Cu2+ ions (Fig. 5d). This indicated that the bound thiol moieties should be reasonable for their high binding affinity towards Cd2+. Pearson’s hard and soft acid and base (HSAB) theory can Fig. 4 Sensing performance of the GSH-SPCE and bare SPCE in the elucidate the phenomenon whereby soft bases (e.g. RSH), simultaneous detection of Cd2+, Pb2+, and Cu2+ with varying concen- rather than hard bases (e.g. RCOOH), have a stronger affinity trations (from 0 to 200 ppb) but a fixed ratio of 1 : 1 : 1. Calibration for soft acids (e.g. Cd2+).63 Vice versa, compared to Pb2+ and curves of the GSH-SPCE and bare SPCE to (a) Cd2+, (b) Pb2+, and (c) Cu2+ as intermediate acids,64 Cd2+ as a soft acid presented a Cu2+. (d) Corresponding voltammograms of GSH-SPCE detecting for a better affinity to soft bases, like thiols. Typical examples are blank sample to 200 ppb mixed solutions (from black to brown). summarized in Table S7.† Therefore, the free thiol groups in the GSH provided potent coordination sites for Cd2+ during the deposition step and thus, increased the concentration of Cd2+ on the electrode surface, even under the interference of Pb2+ and Cu2+, which resulted in the selective enhancement of the Cd2+ sensitivity. Moreover, the defects in the GSH nanosheets structures (as can be seen in the relatively high ID/IG in Fig. S2†) could provide active binding sites for HM ions on the graphene back- bones, which could reduce the ions’ competition and facilitate the deposition of Cd2+ on the working electrode.65 3.3 Interference study of GSH-SPCE and its validation with real samples To mimic real samples, the interference of other ions (i.e. Na+, K+, Ca2+, Mg2+, Ni2+, Zn2+, As3+ and Hg2+ with the 5-fold con- centration of Cd2+, Pb2+, and Cu2+) was studied using the GSH-SPCE. The responses of Cd2+, Pb2+, and Cu2+ decreased to Fig. 5 Reproducibility study and mechanism discussion. The sensi- tivities to (a) Cd2+, (b) Pb2+ and (c) Cu2+ of different GSH-SPCEs and 74%, 97%, and 89%, respectively, compared to those without different bare SPCEs in reproducibility study. (d) The calibration curves interference (Fig. S12†). The signal loss of Cd2+ could be of Cd2+ in simultaneous detection using GSH-SPCEs with comparison caused by Hg2+, which is also a soft acid competing for the to other graphene derivatives modified SPCEs. thiol active sites. GSH-SPCE was then validated with tap water with spiked HMs (Cd : Pb : Cu = 1 : 1 : 1) from 10 ppb to 200 ppb (Fig. S13†). between the GSH and bare groups (Fig. 5(a–c)). GSH was The obtained sensitivity values of Cd2+, Pb2+, and Cu2+ were proven to enhance the sensitivities of the bare SPCEs towards close to the values in the standard solutions (Table S3†). Cd2+ statistically at a highly significant level ( p = 0.0005).33 Afterwards, a recovery/accuracy test was conducted using the Intriguingly, however, the sensitivities to Pb2+ and Cu2+ did GSH-SPCE in spiked tap water containing 90 ppb of Cd2+, not show any significant difference. This finding corroborated Cu2+, and Pb2+. All the recoveries were above 80% as shown in the high specific affinity between GSH and Cd2+ amongst Table S8,† where the reference data from ICP-MS is also these HMs. shown. Integrating filtration membranes or adding mask To understand whether the selective enhancement of the reagents in the fluidic sensing system could further improve Cd2+ sensitivity originated from the thiol moieties or the gra- the accuracy of untreated real samples from a practical point 1654 | Green Chem., 2023, 25, 1647–1657 This journal is © The Royal Society of Chemistry 2023 View Article Online Green Chemistry Paper of view. Modifying the anti-fouling materials on electrodes routes, with a comparative repeatability of Pb2+ and Cu2+, even could be another option and needs to be solved in the future. though the relatively low repeatability of Cd2+ could limit their practical use in highly precise detection. Last, the in situ and 3.4 Comparison with other reported studies automatic testing system, including the reusable GSH-SPCE, This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Last, the GSH-SPCE was compared to other studies, as shown was validated with spiked tap water. Importantly, unlike con- in Table S9.† Although the LODs of the GSH-SPCE (15, 11, and ventional strategies involving metallic additives that can cause 6 ppb for Cd2+, Pb2+, and Cu2+, respectively) were not as low as secondary pollution and have limited reusability, our approach those of the GCEs (sub-ppb) and some bismuth-based SPCEs, offers a metal-free and robust sensing platform. Versatile there are several advantages of the GSH-SPCE to note. functionalization with other chemical groups can be designed First, the metal-free GSH-SPCE is considerably more sus- to construct selective electrodes to target certain analytes in Open Access Article. Published on 07 February 2023. Downloaded on 1/26/2024 4:30:33 PM. tainable and economical to depress mutual interferences. the future. Unlike the traditional strategy, the synthesis of GSH does not involve any metallic precursor and it can be easily prepared in a mass-productive way owing to its wet-chemical synthesis, which matches the large manufacture of SPCEs. Particularly, Author contributions the graphite fluoride (GF) used in the synthesis of the GSH is a Q. Y.: conceptualization, methodology, investigation, data cura- cheap industrial solid lubricant and is easily accessible. tion, visualization, writing–original draft preparation, and Besides, the covalent bond (between amino groups in writing–review & editing; E. P. N.: conceptualization, method- cysteamine and carbon atoms on the graphene backbone) ology, investigation, supervision, writing–original draft prepa- gives GSH extremely high stability in the condition of a largely ration, and writing–review & editing; D. P.: methodology, inves- negative potential and highly acidic solution, which is necess- tigation, visualization, and writing–review & editing; V. Š.: ary for HM detection. These functionalized moieties could be methodology, investigation, visualization, and writing–review maintained on GSH without degradation or deformation in & editing; V. H.: methodology, investigation, visualization, and the continuous cycling measurements. The reusable cycle (23 writing–review & editing; G. R.: investigation and writing– times) surpassed other studies using non-covalently functiona- review & editing; C. C. C. S: investigation and writing–review & lized graphene (1–10 times) and most bismuth-modified editing; A. B.: supervision and writing–review & editing; M. O.: SPCEs (5–16 times, Table S5†). supervision and writing–review & editing; A. M.: conceptualiz- Additionally, our testing system could perform in situ and ation, supervision and writing–review & editing. automatic measurements, allowing the scenario of monitoring drinking water quality at home. In the future, SPCEs modified with various moieties could be achieved via our approach uti- lizing the covalent interaction between the amines of versatile Conflicts of interest molecules and FG. New graphene derivatives with specific moi- There are no conflicts to declare. eties can be designed and synthesized according to their binding affinity to certain HM pollutants to reduce mutual interference. Acknowledgements Conclusion Grant MAT2017-87202-P funded by MCIN/AEI/ 10.13039/ 501100011033. ICN2 is funded by CERCA programme, Sustainable GSH (graphene covalently functionalized with free Generalitat de Catalunya. Grant SEV-2017-0706 funded by thiol groups) was synthesized and modified on SPCEs in order MCIN/AEI/ 10.13039/501100011033. Q. Y. thanks the funding of to alleviate mutual interference. GSH was proven to be able to the Chinese scholarship council. D. P., V. Š. and V. H. thank the boost the diminished sensitivity of the SPCE to Cd2+ from Pb2+ Internal Student Grant Agency of the Palacký University and Cu2+. The Cd2+ sensitivities of GSH-SPCEs demonstrated a (IGA_PrF_2022_019). C.C.C.S. acknowledges funding through significant difference from those of bare SPCEs, while the sen- CAPES – PRINT (Programa Institucional de Internacionalização; sitivities to Pb2+ and Cu2+ were not influenced. The selective grant #88887.310281/2018-00 and 88887.467442/2019-00) and enhancement to Cd2+ originated from the free thiol groups in Mackpesquisa-UPM. A. B. and M. O. acknowledge support by the GSH, according to the best Cd2+ sensitivity in the compara- the project Nano4Future (no. CZ.02.1.01/0.0/0.0/16_019/ tive tests of GA and rGO. This phenomenon can be explained 0000754) financed from the ERDF and ESF. M. O. acknowledges by the HSAB theory, whereby the thiol (soft base) had a good the ERC grant 2D-CHEM, No 683024 from H2020. We appreciate affinity to Cd2+ (soft acid). In multi-HMs simultaneous detec- Ignacio Villarroya from Quimica Analysis Service in UAB for tion, the GSH-SPCE could detect Cd2+, Pb2+, and Cu2+ at up to ICP-MS analysis, Kateřina Roháčová from CATRIN-RCPTM for 15, 11, and 6 ppb, respectively. The high reusability (23 times), the Raman measurement, Tomáš Steklý from CATRIN-RCPTM owing to the covalent functionalization mode, outperforms the for contributing to the synthesis of the GSH, and Vernalyn state-of-the-art SPCEs based on non-covalent functionalization Abarintos from ICN2 for the grammar polishing. This journal is © The Royal Society of Chemistry 2023 Green Chem., 2023, 25, 1647–1657 | 1655 View Article Online Paper Green Chemistry References T. Knutz, E. De Vito-Francesco, R. Allabashi and A. Merkoçi, ACS ES&T Water, 2021, 1, 2459–2555. 1 M. Csuros, Environmental Sampling and Analysis for 28 X. Zhu, B. Liu, H. Hou, Z. Huang, K. M. Zeinu, L. Huang, Technicians, CRC Press, 1st edn, 1994. X. Yuan, D. Guo, J. Hu and J. 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